CD137信号调控内皮细胞间质转化促进小鼠心脑血管新生

doi:10.12300/j.issn.1674-5817.2020.210

摘要/Abstract

摘要： 目的探讨CD137-CD137L信号（简称CD137信号）是否通过调控内皮细胞间质转化（endothelium-mesenchymal transition,End-MT）促进心脑血管新生。方法将小鼠脑微血管内皮细胞（bEnd.3）或小鼠胸主动脉环分为3组,即对照组[10 ng/mL的肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）]、CD137刺激组（10 ng/mL 的TNF-α和5 mg/L的CD137L激动型抗体）和CD137抑制组（200 nmol/L的IWR-1预处理细胞30 min后,再加入10 ng/mL的TNF-α和5 mg/L的CD137L激动型抗体）,以CD137L重组蛋白激动CD137信号。采用荧光定量PCR及蛋白质印迹法分别检测脑微血管内皮细胞中CD31、血管内皮细胞钙黏蛋白（vascular endothelial cadherin,VE-Cadherin）、肌动蛋白α（α-smooth muscle actin,α-SMA）、成纤维细胞特异性蛋白-1（fibroblast specific protein-1,FSP-1）和Wnt蛋白的表达;采用Transwell迁移实验检测穿膜细胞数目,比较细胞迁移能力;内皮细胞管腔形成实验检测各组小鼠脑微血管内皮细胞的管腔形成能力。动脉环血管新生实验检测各组小鼠胸主动脉环新生微血管的形成能力。结果小鼠脑微血管内皮细胞CD137刺激组的CD31、VE-Cadherin mRNA及蛋白表达水平均低于对照组（P<0.05）,FSP-1、Wnt和α-SMA mRNA及蛋白表达表达水平均高于对照组（P<0.05）;CD137抑制组的CD31、VE-Cadherin mRNA及蛋白表达水平均高于CD137刺激组（P<0.05）,FSP-1、Wnt和α-SMA mRNA及蛋白表达水平均低于CD137刺激组（P<0.05）。CD137刺激组的小鼠脑微血管内皮细胞迁移数量多于对照组（P<0.05）,CD137抑制组的细胞迁移数量少于CD137刺激组（P<0.05）。CD137刺激组的小鼠脑微血管内皮细胞小管长度及分支数量均大于对照组（P<0.05）,CD137抑制组的细胞小管长度及分支数均小于CD137刺激组（P<0.05）。CD137刺激组小鼠胸主动脉环的新生微血管数量多于对照组（P<0.05）,CD137抑制组的新生微血管数量少于CD137刺激组（P<0.05）。结论 CD137信号可能通过调控End-MT促进小鼠血管新生。

关键词: 动脉粥样硬化, 血管新生, 内皮细胞间质转化, CD137, Wnt, 小鼠

Abstract: Objective To explore whether CD137-CD137L signaling can promote cardio-cerebral angiogenesis in atherosclerotic plaques via endothelium-mesenchymal transition (End-MT). Methods Mouse brain-derived endothelial cells (Bend.3) and aortas from male mice were divided into the following groups: control (10 ng/mL TNF-α), CD137-activated (10 ng/mL TNF-α + 5 mg/L CD137L agonist antibody), and CD137-inhibited (pretreated with 200 nmol/L IWR-1 for 30 min + 10 ng/mL TNF-α + 5 mg/L CD137L agonist antibody) groups. CD137L agonist antibody was used to activate CD137-CD137L signaling. Fluorescent quantitative PCR was used to determine the expression of CD31, vascular endothelial cadherin (VE-cadherin), fibroblast-specific protein-1F (FSP-1), Wnt, and α-smooth muscle actin (α-SMA) at the mRNA level. Western blotting was used to determine the expression of CD31, VE-cadherin, FSP-1, Wnt, and α-SMA at the protein level. A Transwell assay was used to observe the migration ability of endothelial cells. Matrigel tube formation ability of endothelial cells and mouse aortic ring angiogenesis assay were tested to detect the ability of angiogenesis. Results The expressions of CD31 and VE-cadherin at the mRNA levels in endothelial cells were significantly lower in the CD137-activated group than those in the control group, while the expression was significantly downregulated in the CD137-inhibited group. The expressions of FSP-1, Wnt, and α-SMA were opposite (all P < 0.05). The expressions of CD31 and VE-cadherin proteins in endothelial cells were significantly higher in the CD137-activated group than those in the control group, while the expression was significantly downregulated in the CD137 inhibited group. The expressions of FSP-1, Wnt, and α-SMA were opposite (all P < 0.05). Migration cell number was markably higher in the CD137-activated group than that in the control group, while it was significantly lower in the CD137-inhibited group than that in the control group (P < 0.05). The values of the formation of the tube length (P < 0.05) and branch number (P < 0.05) were both significantly higher in the CD137-activated group than those in the control group, while these values were significantly lower in the CD137-inhibited group than those in the control group (P < 0.05). The number of microvessel outgrowths in the aortic rings was dramatically increased in the CD137-activated group than that in the control group, while it was reduced in the CD137-inhibited group (P < 0.05). Conclusion CD137-CD137L signaling can promote cardio-cerebral angiogenesis in atherosclerotic plaques by activating End-MT.

Key words: Atherosclerosis, Angiogenesis, Endothelium-mesenchymal transition, CD137, Wnt, Mice

中图分类号:

Q95-33
R-332

翁嘉懿, 马雪兴, 李渊, 孙康云. CD137信号调控内皮细胞间质转化促进小鼠心脑血管新生[J]. 实验动物与比较医学, 2021, 41(5): 418-425.

WENG Jiayi, MA Xuexing, LI Yuan, SUN Kangyun. CD137 Signaling Promotes Cardio-cerebral Angiogenesis in Mice Through Regulating Endothelial-mesenchymal Transition[J]. Laboratory Animal and Comparative Medicine, 2021, 41(5): 418-425.

图/表 3

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